Different pathways are likely to regulate the initiation of DNA replication. For example, the post-translational modification of the proteins required for synthesis are known to be pivotal in intricate ways.sup.1-5. Interestingly, proteins that bind to origins of replication also function in the control of transcription.sup.6,7. The roles of transcription factors in regulating chromosomal replication are ambiguous. However, numerous experiments have shown that tissue-specific gene expression is correlated with early replication of the active gene and its flanking DNA, while the same gene when inactive in another tissue replicates late in the cell cycle.sup.8. This implies a link between transcription control and replication control.
Bovine Papilloma Virus type 1 (BPV-1 ) provides a framework for exploring the roles of transcription factors in eukaryotic DNA replication. In transformed cells, the viral chromosome is maintained as a stable nuclear plasmid replicating in synchrony with the host DNA. Two vital proteins, E1 and E2, are both necessary and sufficient for replication.sup.9. E1 is a 68 kD protein and vital DNA with mutations in this ATP binding protein cannot be maintained as nuclear plasmids.sup.10. Three related site-specific DNA binding proteins are encoded by the E2 ORF.sup.1 : a 48 kD transactivator and two represson lacking the activation domain, E2C and E8/E2. The 48 kD transactivator binds to DNA as a dimer, and in combination with cellular factors including SP-1.sup.12 activates transcription from a number of vital promoters. The relative concentrations of the E2 family of ;proteins thus intricately regulate the transcriptional program of the viral plasmids. Studies from our laboratory showed that the 48 kD E2 protein could form a tight complex with the E1 protein.sup.13. Partially purified E1 displayed a weak specific DNA binding activity, and this activity was markedly stimulated by E2. To facilitate mechanistic studies and to ascertain if E2 plays a direct role in DNA replication, we developed a cell-free replication system.